Search Results (72)

Background: The rapid emergence of antibiotic resistance in Acinetobacter baumannii has led the World Health Organization (WHO) to designate it as a “high priority” pathogen. The emergence of multidrug-resistant (MDR) and pandrug-resistant (PDR) strains poses considerable treatment challenges. As antimicrobial resistance (AMR) escalates toward a post-antibiotic era, innovative therapeutic solutions are urgently needed. Objectives: To clone, over-express, and characterize a novel endolysin, LysTAC1, from Acinetobacter phage TAC1 for its antibacterial efficacy against multidrug-resistant bacteria. Methods: A 24 kDa endolysin featuring a glycoside hydrolase Family 19 chitinase domain was tested against carbapenem-resistant Acinetobacter baumannii clinical isolates and various Escherichia coli strains following outer membrane permeabilization with Ethylenediaminetetraacetic acid (EDTA). Stability assays and molecular docking studies were performed. Results: LysTAC1 demonstrated potent lytic activity against Gram-negative bacteria but showed no activity against Gram-positive bacteria (Staphylococcus aureus ATCC 29213 and Enterococcus gallinarum HCD 28-1). LysTAC1 maintained activity across pH 6–9 and temperatures 4–65 °C, with differential sensitivity to metal ions where K⁺ showed no inhibitory effect at any concentration (0.1–100 mM), and Fe²⁺ was non-inhibitory at lower concentrations (0.1–1 mM), while Mg²⁺ and Ca²⁺ demonstrated concentration-dependent inhibition across the tested range (0.1–100 mM). Molecular docking revealed LysTAC1 interactions with chitinase substrates 4-nitrophenyl N-acetyl-β-D-glucosaminide and 4-nitrophenyl N, N-Diacetyl-β-D-chitobioside, with binding energies of −5.82 and −6.85 kcal/mol, respectively. Conclusions: LysTAC1 shows significant potential as a targeted therapeutic agent against A. baumannii with robust stability under physiological conditions. Full article

Background/Objectives: Listeria monocytogenes is a high-risk pathogen in the food industry involved in several outbreaks. Bacteriocins are natural-origin antimicrobial peptides or proteins that represent a good alternative to synthetic antimicrobials capable of inhibiting the growth of pathogens. This study aimed to purify and identify bacteriocins from the cell-free supernatant of Enterococcus lactis-67, which exhibits antagonistic activity against L. monocytogenes. Methods: Protein purification was performed by precipitation with ammonium sulfate, dialysis, and fast protein liquid chromatography. Active protein fractions were analyzed by SDS-PAGE and identified by mass spectrometry. Results: In addition to enterocins A and B, a novel 47 kDa bacteriocin with LysM and NlpC/P60 domains, on the N- and C-terminal regions, respectively, was identified. This enterocin has not been described for Enterococcus before. Conclusions: This study contributes to the identification of new natural and effective strategies for ensuring food safety. Full article

Endolysins, phage-derived enzymes capable of lysing bacterial cell walls, hold significant promise as novel antimicrobials against resistant Gram-positive and Gram-negative pathogens. In this study, we undertook an integrative approach combining extensive in silico analyses and experimental validation to characterize the novel endolysin LysPALS22. Initially, sixteen endolysin sequences were selected based on documented lytic activity and enzymatic diversity, and subjected to multiple sequence alignment and phylogenetic analysis, which revealed highly conserved catalytic and binding domains, particularly localized to the N-terminal region, underscoring their functional importance. Building upon these sequence insights, we generated three-dimensional structural models using Swiss-Model, EBI-EMBL, and AlphaFold Colab, where comparative evaluation via Ramachandran plots and ERRAT scores identified the Swiss-Model prediction as the highest quality structure, featuring over 90% residues in favored conformations and superior atomic interaction profiles. Leveraging this validated model, molecular docking studies were conducted in PyRx with AutoDock Vina, performing blind docking of key peptidoglycan-derived ligands such as N-Acetylmuramic Acid-L-Alanine, which exhibited the strongest binding affinity (−7.3 kcal/mol), with stable hydrogen bonding to catalytic residues ASP46 and TYR61, indicating precise substrate recognition. Visualization of docking poses using Discovery Studio further confirmed critical hydrophobic and polar interactions stabilizing ligand binding. Subsequent molecular dynamics simulations validated the stability of the LysPALS22–NAM-LA complex, showing minimal structural fluctuations, persistent hydrogen bonding, and favorable interaction energies throughout the 100 ns trajectory. Parallel to computational analyses, LysPALS22 was heterologously expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), where SDS-PAGE and bicinchoninic acid assays validated successful protein production; notably, the P. pastoris-expressed enzyme displayed an increased molecular weight (~45 kDa) consistent with glycosylation, and achieved higher volumetric yields (1.56 ± 0.31 mg/mL) compared to E. coli (1.31 ± 0.16 mg/mL), reflecting advantages of yeast expression for large-scale production. Collectively, these findings provide a robust structural and functional foundation for LysPALS22, highlighting its conserved enzymatic features, specific ligand interactions, and successful recombinant expression, thereby setting the stage for future in vivo antimicrobial efficacy studies and rational engineering efforts aimed at combating multidrug-resistant Gram-negative infections. Full article

Drought is the single largest abiotic threat to soybean yield, yet the lectin genes that mediate drought perception and signaling in this crop have never been systematically mapped. We reanalyzed the public RNA-seq dataset GSE237798 (Williams 82 leaves, 7-day water withdrawal) with an updated fastp–HISAT2–featureCounts–DESeq2 pipeline and a curated catalog of 359 soybean lectin loci. Of the 127 lectin transcripts showing any drought-dependent shift, only 15 were stringently differentially expressed with substantial fold changes: 7 were upregulated and 8 downregulated. These genes span four families, GNA, legume, LysM and Nictaba-related lectins, and are heavily biased toward lectin receptor-like kinases (11 of 15), pinpointing the plasma membrane as the main control node. Gene Ontology enrichment highlights protein autophosphorylation and signal-transduction terms, and the inspection of AlphaFold models together with established lectin knowledge indicates that G- and L-type lectin domains have largely lost canonical carbohydrate-binding residues, whereas LysM and Nictaba proteins retain conserved folds compatible with ligand binding. The data expose a focused, modular lectin program rather than the broad activation often assumed: most soybean lectins stay silent under drought conditions, and only a defined subset toggles their expression, albeit mildly. Full article

Microbe–microbe interactions have been explored at the molecular level to a lesser degree than plant–pathogen interactions, primarily due to the economic impact of crop losses caused by pathogenic microorganisms. Effector proteins are well known for their role in disease development in many plant–pathogen pleinteractions, but there is increasing evidence showing their involvement in other types of interaction, including microbe–microbe interactions. Through the use of LC-MS/MS sequencing, effector candidates were identified in the in vitro interaction between a banana pathogen, Pseudocercospora fijiensis and a biological control agent, Trichoderma harzianum. The diverse interaction secretome revealed various glycoside hydrolase families, proteases and oxidoreductases. T. harzianum secreted more proteins in the microbial interaction compared to P. fijiensis, but its presence induced the secretion of more P. fijiensis proteins that were exclusive to the interaction secretome. The interaction secretome, containing 256 proteins, was screened for effector candidates using the algorithms EffHunter and WideEffHunter. Candidates with common fungal effector motifs and domains such as LysM, Cerato-platanin, NPP1 and CFEM, among others, were identified. Homologs of true effectors and virulence factors were found in the interaction secretome of T. harzianum and P. fijiensis. Further characterization revealed a potential novel effector of T. harzianum. Full article

Oleanolic acid derivatives, specifically lactones (2–8) and bromolactones (9–14), were synthesised and evaluated for their cytotoxic, antioxidant, and pharmacokinetic profiles. The compounds were characterised using molecular docking simulations targeting the 1M17 protein, representing the EGFR tyrosine kinase domain. Compound 6 emerged as the most promising candidate, demonstrating strong interactions with residues critical for EGFR activity, such as LYS 721 and ASP 831. Biological assays revealed that compounds 6, 2, and 10 exhibited IC₅₀ values across various cancer cell lines in the micromolar range, with a favourable Selectivity Index. Antioxidant activity assays (CUPRAC and DPPH) highlighted compound 7 as the most substantial electron donor and compound 10 as the most influential radical scavenger. ADMETox analysis confirmed the favourable pharmacokinetic and safety profiles of the derivatives. These findings underscore the potential of the selected oleanolic acid derivatives as drug candidates for targeted cancer therapies, offering cytotoxic and antioxidant benefits. Despite their promising cytotoxic and antioxidant activities, translating oleanolic acid derivatives to clinical applications remains challenging due to their bioavailability and metabolic stability. Our findings highlight compound 6 as a leading candidate with enhanced activity, providing a foundation for further optimising and developing EGFR-targeted anticancer therapies. Full article

Alginate lyases are of great importance in biotechnological and industrial processes, yet research on these enzymes from Mesonia genus bacteria is still limited. In this study, a novel PL6 family alginate lyase, MhAly6, was cloned and characterized from the deep-sea bacterium Mesonia hitae R32. The enzyme, composed of 797 amino acids, contains both PL6 and GH28 catalytic domains. A phylogenetic analysis revealed its classification into subfamily 1 of the PL6 family. MhAly6 showed optimal activity at 45 °C and pH 9.0, retaining over 50% activity after 210 min of incubation at 40 °C, highlighting its remarkable thermal stability. The enzyme exhibited degradation activity toward sodium alginate, Poly M, and Poly G, with the highest affinity for its natural substrate, sodium alginate, producing alginate oligosaccharides (AOSs) with degrees of polymerization (DP) ranging from 2 to 7. Molecular docking identified conserved catalytic sites (Lys241/Arg262) and Ca²⁺ binding sites (Asn202/Glu234/Glu236), while the linker and GH28 domain played an auxiliary role in substrate binding. Antioxidant assays revealed that the MhAly6-derived AOSs showed potent radical-scavenging activity, achieving 80.64% and 95.39% inhibition rates against DPPH and ABTS radicals, respectively. This work not only expands our understanding of alginate lyases from the Mesonia genus but also highlights their biotechnological potential for producing functional AOSs with antioxidant properties, opening new avenues for their applications in food and pharmaceuticals. Full article

The initial phase of an insect’s innate immune response to foreign pathogens is triggered by the identification of exogenous invaders, a mechanism facilitated by pattern recognition receptors. Among these receptors, peptidoglycan recognition proteins (PGRPs), abundant in insects, are essential components of the innate immune system. The roles of PGRPs have been extensively elucidated in Drosophila melanogaster; however, the mechanism underlying the immune response of Aedes albopictus to pathogens is unclear. Herein, we successfully cloned the full-length cDNA of a PGRP gene from Ae. albopictus, designated as the AaPGRP-LB gene. The open reading frame of AaPGRP-LB encodes 203 amino acids, including a secretion signal peptide and a canonical PGRP conserved domain. Multisequence alignment revealed that AaPGRP-LB possesses the amino acid residues essential for zinc binding and amidase activity. Molecular docking studies demonstrated that AaPGRP-LB exhibits a strong binding affinity for DAP-type and LYS-type peptidoglycan. The mRNA expression level of the AaPGRP-LB gene significantly increased after oral infection with Escherichia coli or Staphylococcus aureus. The purified recombinant AaPGRP-LB (rAaPGRP-LB) exhibited strong agglutination properties and demonstrated significant antimicrobial efficacy against E. coli and S. aureus in the presence of zinc ions. This study highlights the critical role of AaPGRP-LB in the immune response of Ae. albopictus. These findings provide a foundation for future research on mosquito immune pathways for innovative vector control and disease prevention strategies. Full article

Arbuscular mycorrhizal (AM) symbiosis, a mutually beneficial interaction between plant roots and AM fungi, plays a key role in plant growth, nutrient acquisition, and stress tolerance, which make it a major focus for sustainable agricultural strategies. This intricate association involves extensive transcriptional reprogramming in host plant cells during the formation of arbuscules, which are specialized fungal structures for nutrient exchange. The symbiosis is initiated by molecular signaling pathways triggered by fungal chitooligosaccharides and strigolactones released by plant roots, which act as chemoattractants and signaling molecules to promote fungal spore germination, colonization, and arbuscule development. Calcium spiking, mediated by LysM domain receptor kinases, serves as a critical second messenger in coordinating fungal infection and intracellular accommodation. GRAS transcription factors are key components that regulate the transcriptional networks necessary for arbuscule development and maintenance, while small RNAs (sRNAs) from both plant and fungi, contribute to modifications in gene expression, including potential bidirectional sRNA exchange to modulate symbiosis. Understanding the molecular mechanisms related to AM symbiosis may provide valuable insights for implementation of strategies related to enhancing plant productivity and resilience. Full article

Background: Long COVID-19 has been characterized by the presence of symptoms lasting longer than 4 weeks after the acute infection. The pathophysiology of clinical manifestations still lacks knowledge. Objective: The objective of this paper was to evaluate metabolite abundance in the saliva of long COVID patients 60 days after hospital discharge. Methods: A convenience sample was composed of 30 post-discharge patients with long COVID and seven non-COVID-19 controls. All COVID-19 patients were evaluated by demographic characteristics, spirometry, 6 min walk test (6mWT), Saint George Respiratory Questionnaire (SGRQ), and body composition. Metabolomics was performed on saliva. Results: The long COVID-19 patients were 60.4 ± 14.3 years-old, and 66% male. Their lean body mass was 30.7 ± 7.3 kg and fat mass, 34.4 ± 13.7 kg. Spirometry evaluation showed forced vital capacity (FVC) of 3.84 ± 0.97 L with 96.0 ± 14.0% of the predicted value, and forced expiratory volume in the first second (FEV₁) of 3.11 ± 0.83 L with 98.0 ± 16.0 of the predicted value. The long COVID-19 patients had reduced maximal inspiratory (90.1 ± 31.6 cmH₂O) and maximal expiratory (97.3 ± 31.0 cmH₂O) pressures. SGRQ showed domain symptoms of 32.3 ± 15.2, domain activities of 41.9 ± 25.6, and domain impact 13.7 ± 11.4, with a mean of 24.3 ± 14.9%. Physical capacity measured by distance covered in the 6mWT was 418.2 ± 130 m with a 73.3% (22.3–98.1) predictive value. The control group consisted of 44.1 ± 10.7-year-old men with a body mass index of 26.5 ± 1.66 Kg/m². Metabolomics revealed 19 differentially expressed metabolites; expression was lower in 16 metabolites, and 2 metabolites were absent in the COVID-19 patients compared to controls. Calenduloside G methyl ester (p = 0.03), Gly Pro Lys (p = 0.0001), and creatine (p = 0.0001) expressions were lower in patients than controls. Conclusions: Long COVID-19 patients present less abundance of calenduloside G methyl ester, Gly Pro Lys, and creatine in saliva than healthy controls. Lower creatine abundance may be related to reduced physical capacity and fatigue Full article

Three-finger proteins (TFPs), or Ly6/uPAR proteins, are characterized by the beta-structural LU domain containing three protruding “fingers” and stabilized by four conserved disulfide bonds. TFPs were initially characterized as snake alpha-neurotoxins, but later many studies showed their regulatory roles in different organisms. Despite a known expression of TFPs in vertebrates, they are poorly studied in other taxa. The presence of TFPs in starfish was previously shown, but their targets and functional role still remain unknown. Here, we analyzed expression, target, and possible function of the Lystar5 protein from the Asterias rubens starfish using bioinformatics, qPCR, and immunoassay. First, the presence of Lystar5 homologues in all classes of echinoderms was demonstrated. qPCR revealed that mRNA of Lystar5 and LyAr2 are expressed mainly in coelomocytes and coelomic epithelium of Asterias, while mRNA of other TFPs, LyAr3, LyAr4, and LyAr5, were also found in a starfish body wall. Using anti-Lystar5 serum from mice immunized by a recombinant Lystar5, we confirmed that this protein is expressed on the surface of coelomocytes and coelomic epithelium cells. According to ELISA, a recombinant analogue of Lystar5 bound to the membrane fraction of coelomocytes and coelomic epithelium but not to the body wall or starfish arm tip. Analysis by LC-MALDI MS/MS suggested integrin α-8-like protein expressed in the coelomocytes and coelomic epithelium as a target of Lystar5. Thus, our insights propose the important role of TFPs in regulation of starfish physiology and show prospects for their further research. Full article

A possible molecular mechanism of the ligand–receptor binding of Ac-Lys-Lys-Lys-NH₂ (Ac-KKK-NH₂) to the Na_V1.8 channel that is responsible for nociceptive signal coding in the peripheral nervous system is investigated by a number of experimental and theoretical techniques. Upon Ac-KKK-NH₂ application at 100 nM, a significant decrease in the effective charge carried by the Na_V1.8 channel activation gating system Z_eff is demonstrated in the patch-clamp experiments. A strong Ac-KKK-NH₂ analgesic effect at both the spinal and supraspinal levels is detected in vivo in the formalin test. The distances between the positively charged amino groups in the Ac-KKK-NH₂ molecule upon binding to the Na_V1.8 channel are 11–12 Å, as revealed by the conformational analysis. The blind docking with the Na_V1.8 channel has made it possible to locate the Ac-KKK-NH₂ binding site on the extracellular side of the voltage-sensing domain VSD_I. The Ac-KKK-NH₂ amino groups are shown to form ionic bonds with Asp151 and Glu157 and a hydrogen bond with Thr161, which affects the coordinated movement of the voltage sensor up and down, thus modulating the Z_eff value. According to the results presented, Ac-KKK-NH₂ is a promising candidate for the role of an analgesic medicinal substance that can be applied for pain relief in humans. Full article

Pseudocercospora (previously Mycosphaerella) fijiensis is a hemibiotroph fungus and the causal agent of black Sigatoka disease, one of the most significant threats to banana production worldwide. Only a few genomics reports have paid any attention to effector proteins, which are key players in pathogenicity. These reports focus on canonical effectors: small secreted proteins, rich in cysteines, containing a signal peptide and no transmembrane domain. Thus, bias in previous reports has resulted in the non-canonical effectors being, in effect, excluded from the discussion of effectors in P. fijiensis pathogenicity. Here, using WideEffHunter and EffHunter, bioinformatic tools which identify non-canonical and canonical effectors, respectively, we predict, for the first time, the full effectorome of P. fijiensis. This complete effectorome comprises 5179 proteins: 240 canonical and 4939 non-canonical effectors. Protein families related to key functions of the hemibiotrophic lifestyle, such as Salicylate hydroxylase and Isochorismatase, are widely represented families of effectors in the P. fijiensis genome. An analysis of the gene distribution in core and dispensable scaffolds of both classes of effectors revealed a novel genomic structure of the effectorome. The majority of the effectors (canonical and non-canonical) were found to be harbored in the core scaffolds, while dispensable scaffolds harbored less than 10% of the effectors, all of which were non-canonical. Additionally, we found the motifs RXLR, YFWxC, LysM, EAR, [Li]xAR, PDI, CRN, and ToxA in the effectors of P. fijiensis. This novel genomic structure of effectors (more enriched in the core than in the dispensable genome), as well as the occurrence of effector motifs which were also observed in four other fungi, evidences that these phenomena are not unique to P. fijiensis; rather, they are widely occurring characteristics of effectors in other fungi. Full article

Acid-sensing ion channels (ASICs), which act as proton-gating sodium channels, have garnered attention as pharmacological targets. ASIC1a isoform, notably prevalent in the central nervous system, plays an important role in synaptic plasticity, anxiety, neurodegeneration, etc. In the peripheral nervous system, ASIC1a shares prominence with ASIC3, the latter well established for its involvement in pain signaling, mechanical sensitivity, and inflammatory hyperalgesia. However, the precise contributions of ASIC1a in peripheral functions necessitate thorough investigation. To dissect the specific roles of ASICs, peptide ligands capable of modulating these channels serve as indispensable tools. Employing molecular modeling, we designed the peptide targeting ASIC1a channel from the sea anemone peptide Ugr9-1, originally targeting ASIC3. This peptide (A23K) retained an inhibitory effect on ASIC3 (IC₅₀ 9.39 µM) and exhibited an additional inhibitory effect on ASIC1a (IC₅₀ 6.72 µM) in electrophysiological experiments. A crucial interaction between the Lys23 residue of the A23K peptide and the Asp355 residue in the thumb domain of the ASIC1a channel predicted by molecular modeling was confirmed by site-directed mutagenesis of the channel. However, A23K peptide revealed a significant decrease in or loss of analgesic properties when compared to the wild-type Ugr9-1. In summary, using A23K, we show that negative modulation of the ASIC1a channel in the peripheral nervous system can compromise the efficacy of an analgesic drug. These results provide a compelling illustration of the complex balance required when developing peripheral pain treatments targeting ASICs. Full article

Tinea cruris, a dermatophyte fungal infection predominantly caused by Trichophyton rubrum and Epidermophyton floccosum, primarily affects the groin, pubic region, and adjacent thigh. Its recurrence is frequent, attributable to repeated fungal infections in susceptible individuals, especially those with onychomycosis or tinea pedis, which act as reservoirs for dermatophytes. Given the persistent nature of tinea cruris, vaccination emerges as a promising strategy for fungal infection management, offering targeted, durable protection against various fungal species. Vaccines stimulate both humoral and cell-mediated immunity and are administered prophylactically to prevent infections while minimizing the risk of antifungal resistance development. Developing fungal vaccines is challenging due to the thick fungal cell wall, similarities between fungal and human cells, antigenic variation, and evolutionary resemblance to animals, complicating non-toxic target identification and T-cell response variability. No prior research has shown an mRNA vaccine for T. rubrum. Hence, this study proposes a novel mRNA-based vaccine for tinea cruris, potentially offering long-term immunity and reducing reliance on antifungal medications. This study explores the complete proteome of T. rubrum, identifying potential protein candidates for vaccine development through reverse vaccinology. Immunogenic epitopes from these candidates were mapped and integrated into multitope vaccines and reverse translated to construct mRNA vaccines. Then, the mRNA was translated and computationally assessed for physicochemical, chemical, and immunological attributes. Notably, 1,3-beta-glucanosyltransferase, CFEM domain-containing protein, cell wall galactomannoprotein, and LysM domain-containing protein emerged as promising vaccine targets. Antigenic, immunogenic, non-toxic, and non-allergenic cytotoxic T lymphocyte, helper T lymphocyte, and B lymphocyte epitopes were selected and linked with appropriate linkers and Toll-like receptor (TLR) agonist adjuvants to formulate vaccine candidates targeting T. rubrum. The protein-based vaccines underwent reverse translation to construct the mRNA vaccines, which, after inoculation, were translated again by host ribosomes to work as potential components for triggering the immune response. After that, molecular docking, normal mode analysis, and molecular dynamic simulation confirmed strong binding affinities and stable complexes between vaccines and TLR receptors. Furthermore, immune simulations of vaccines with and without adjuvant demonstrated activation of immune responses, evidenced by elevated levels of IgG1, IgG2, IgM antibodies, cytokines, and interleukins. There was no significant change in antibody production between vaccines with and without adjuvants, but adjuvants are crucial for activating the innate immune response via TLRs. Although mRNA vaccines hold promise against fungal infections, further research is essential to assess their safety and efficacy. Experimental validation is crucial for evaluating their immunogenicity, effectiveness, and safety. Full article